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Effects of Cortisol on Serially Propagated Fibroblast Cell Cultures Derived from the Rabbit Fetal Lung and skin' BARRYT. SMITH'AND C. J. P. ~ J I R O U D ~ Mc (;iil Unierersip, Montreal CImiidren'sHospital Rpseurch Itzstitlate, Montreul, Quebec H31f IP3 Keceived May 20, 1975 SMITH, B. T.. and GIROUD,C. J. P. 1975. Effects of ort ti sol on serially propogated fibroblast cell cultures derived from the rabbit fetal lung and skin. Can. J . Physiol. Pharmacol. 53, 10371041. Corti301 affects the growth of serially propogated, fibroblast cell cultures derived from the rabbit fetal lung in a manner which is dependent upon the gestational age of the material used: early in gestation (20 days), the hormone (10-7-10-5 M) stimulates [6-3Hlthymidine incorporaand 10 M) inhibits this process. tion into DNA, while in late gestation (28 days), cortisol M) at both gestational Cultures derived from the rabbit fetal skin are inhibited by cortisol apes. Fibroblasts derived from lung, but not from skin, efficiently convert cortisone to cortisol and this activity increases with advancing gestation. Cortisol does not affect the incorporation of [7H]cholineinto lecithin by confluent cult~aresof any of the fibroblast types studied.
Introduction The fibroblast is a cell type found in most nlammalian tissues whose main function is considered to be the generation of stromal support. Ht has long been known that fibroblasts cultured in vitru retain, and in many cases reflect, the genetic endowment of their source, allowing for the study and diagnosis of a variety of genetic disorders. Evidence that fibroblasts derived from different organs of the same donor (Martin et al. 1970), or from different anatomical sites on the same organ (Pinsky et al. 1972; Mulay et al. 1972) posscss different functions is more limited. Also, there is only limited evidence to suggest that fibroblasts derived from the same source at different stages of development could exhibit changing in vsro phenotypic expressions (Maciera-Coelho and Ponten 1969; Martin et al. 1970). W e have previously shown that mixed primary cell cultures (including fibroblasts and epithelial elements) from the rabbit fetal lung respond differently to cortisol depending upon the stage of gestation at which they are isolated: early in gestation (20-22 days) the hormone stimulates cellular growth, 'late in gestation (28
days) growth is inhibited while the incorporation of labelled precursors into lecithin is significantly cnhanced (Smith et al. 1974). The present studies were designed to determine whether serially propogated fibroblast cell cultures derived from the fetal lung and skin at different gestational ages (20 and 28 days) would reflect gestation-dependent changes in metabolic activities and responses to cortisol. It was also our aim to determine, by inference, which of the responses to cortisol noted in mixed cultures could be accounted for by the fibroblasts present.
'Supported by the Medical Research Council of Canada. Tellow, Medical Research Council of Canada. Present address, to which reprint requests should be sent: Department of Paediatrics, Queen's University, Kingston, Ontario K7L 3N6. 3Associate, Medical Research Council of Canada.
Biological Pregnant white New Zealand rabbits were obtained from Canadian Breeding Farnas, St. Constant, Que., and killed at 20 o r 28 days gestation with a lethal intravenous dose of pentobarbital. The fetuses were removed immediately to provide tissues for the initiation of cell lines.
Methods Culture media used in this study consisted of Hana's
F 10 medium (Difco, Detroit, Mich.) supplemented with 10% fetal calf serum (Flow Laboratories, Rockville, Md.) and antibiotics (Smith et al. 1974). The followillg radioactive compounds were Furchased from New England Nuclear, Boston, Mass.: [3HH]TdR' (sa 9 Ci/mnaol) , [Me-'Hlcholine chloride (sa 2.34 Ci/mn~ol), [4-'4C~ortisol (sa 55.3 mCi/ mmol ) , and [l ,2-3H]cortisone (sa 40 Ci/rnmol) . Radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrophotometer, model 4322, with an efficiency of 27% for TI under single label counting conditions, and of 9.7% and 48% for 3H and 14C, respectively, under double Babel conditions.
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CAN. I . PHYSHOL,. PHARMACOL. VOL,. 53, 1995
The fetal lungs were removed, washed, minced, and trypsinized into a dispersed cell population as previously described (Smith et ul. 1974). The dispersed, mixed cell populations were placed in 30-mm plastic petri dishes (Falcon). Pure fibroblastic cell cultures, as judged by careful phase contrast microscopy, were obtained simply by changing the media 2 k after implantation, at which time only fibroblasts had adhered to the ciishes (Crystal, R.G.: personal cornm~anicakion). Fetal skin fibroblast cell cultures were initiated from I rnm explants by standard techniques. In ali cases, explants were taken from the upper, anterior abdominal wall just below the xiphisternal junction. The culte~res were maintained in 30-mm plastic petri dishes containing 3 rnB of medium and maintained in an atmosphere s f 5% @OLand high humidity at 37 "C. The media were changed every 48-32 h. The cells were transferred by treatment with buffered trypsin-EDTA solution (containing 0.5 g of trypsin, 1 :250, and 2.0 g of EDTA per litre. Grand Island Biological Co., Grand Island, N . Y . ) with a 1 : 3 split every 7-10 days. All studies to be reported here were carried out between the fourth and sixth passage levels. E,+cr 0f &Tortiso/ on Cell G a o ~ ~ t h The effect of cortisol on cellular growth was determined by the incorporation of [WH]TdR into DNA. Cells were plated at 2 X 10' per dish and grown for 48 h in control media. They were then exposed for 24 h to various concentrations ( lQ-0-10-5 M ) of cortisol in control nmedium, following which they were incubated for 6 h with [SHJTdW (2 pCi) in 3 rnl of serum-free medium. Following the incubation, the media were decanted and the cultures washed with three rinses of ice-cold 0.9% saline. The cells were scraped from the dishes, sonicated in 2 ram1 of saline, and the DNA precipitated by the addition of 2 rani of 20% trichlosoacetic acid. Aliquots of the precipitate were taken for counting and the remainder was used for the determination of DNA content by the diphenylamine colorimetric reaction (Burton 1956), using 2-deoxyribose as standard. Results are expressed as counts per minute of "H per nanomole of DNA. Effect of Cortisol OPT ["HICholine Inc~rjloratiot~ into Lecl'thi~z Cultures were grown to confluence in media containing varying concentrations ( l O - O - I O - W M ) of cortisol. Under these conditions, the DNA content of confluent cultures was not affected by the hormone. The cultures were then incubated for 6 h with serumfree media containing 4 X 10" dpna (1.5 nmsH) of VHIcholine. Following the incubation, the media were dec,mted and the c~mltureswashed with three changes of ice-cold saline. The cellular material Mias then scraped from the dishes in three changes (total 5.0 ml) of methanol. Ten millilitres sf chloroform were added and the n~ixturewas tightly capped and left overnight at room temperature. Five anillilitres of
"Abbreviation : [3H]TdW, 66-%]thymidine.
modified aqueous phase for the Folch extraction (distilled water with 100 mA4 KC1 and 10 mA4 choline) (Folch et ral. 1957; Mason ct a / . 1972) were then added. The mixture was shaken and placed at -20 " C to aid phase separation. The lower (organic) phase was filtered and evaporated to dryness. The lipid extracts were then processed for the chromatographic isolation of lecithin as previously described (Smith et al. 1974). The lecithin fractions were counted for their content of 'Hp and the results expressed as picomoles of ["Hlcholine incorporated into lecithin per dish. Cortisone-Cortisol In terconversioaa,~ The ability of the cells to interconvert cortisone and cortisol ( I l-oxidoreductase activity) was studied by inctabating confluent cultures with a high, equimolar concentration (2 X 18-"1 of [14CCortrtisol (2 >: 10" dpm) and [3H]cortisone ("I 110" dprn). The media were then removed and extracted with three volumes of ethyl acetate. The ethyl acetate extracts were processed for the isolation and purification, by paper chromatography, of cortisol and cartisone as previously described (Smith et aE. 1 9 7 3 ) . The incubated radioactivity (%I and 14C) was always recovered to the extent of 75%. The content of 3H and 'C in the cortisol and cortisone fractions was determined and the percentage conversion of cortisol to cortisone and of cortisone to cortisol was calculated. Since the conversion of cortisol to cortisone results in biological inactivation of the hormone while the reverse results in biological activation (Berliner and Dougherty I961 ), the percentage conversion sf cortisone to cortisol was arbitrarily assigned a positive sign while the percentage conversion of cortisol to cortisone was given a negative sign. The two values were then arithmetically summed to give 'net activation'. That is, positive valiaes indicate the net conversion of inactive to active hormone, while negative values reflect net inactivation. StutisticaI All values reported represent the mean (r+-SD)of triplicate observations. Statistical significance of observed differences was assessed using Student's t test for unpaired data.
Results Eflec-t of Cortisol on Cell Growth Addition of cortisol to the cultbarc media of serially propagated fetal fibroblast cell cultures affects the incorporation of I3H]Tc%R4 illto acidinsoluble material irm a manner which is strongly influenced by gestational agc and organ of origin (Table 1) . Lung fibroblasts are most sensitive to the effects of cortisol: ["HITdR incorporation by 20-day lung fibroblasts is stimulated by cortisol. at cormcentrations as low as 10 - 7 M ( p < 0.005), while this process is i~~hibitcd in the case sf 28-day lung fibroblasts,
3 039
SMITH AND GIMBUD: CORTISOI, EFFECTS ON FIBMBBLAS'T CEI,L CULTURES
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TABLE 1. Effect of cortisol on DNA synthesisa by fetal rabbit fibroblast cell cultures
Cell type Cortisol (M)
Lung (20 days)
Skin (20 days)
Lung (28 days)
Skin (28 days)
OAs measured by the incorporation of [3H]thyrnidine. Each value represents the mean ( + SD) counts per minute of 3H recovered in the acidinsoluble fraction per nanomoles of DNA. bp values for statistically significant diKerences (us. control for each cell type) are sho\sn in parentheses.
TABLE 2. Lack of effect of cortisol on lecithin synthesisa by fetal rabbit fibroblast cell cultures
Cel I type Cortisol (Mb
Lung (20 days)
Lung (28 days)
Skin (20 days)
Skin (28 days)
OEach value represents the mean ( 9 SD) picornoles of [311]choline incorpetrated into lecithin per culture dish.
at doses of IV ( p < 0.OS), and 10--Q:W ( p < 0.02). In the case of fetal rabbit skin fibroblasts, cortisol affects [:%I]TdW incorporation only at the highest concentration studied (10- "V), negatively in cultures from both gestations (20 days, g < 0.02; 28 days, p < 0.05). Further studies will be needed to determine if these effects of cortisol on cell growth depend upon the presence of fetal calf serum.
dominantly reduce cortisone to its active form, cortisol. This activity is significantly greater in cells derived from the 28-day lung as compared to the 20-day lung (15.6 0.6 vs. 3.5 t 44.7, p < 0.001 ) . In contrast, fetal skin fibroblasts predonsinantly oxidize cortisol to cortisone, to a similar extent at both gestational ages.
*
Discussion Our results show that serially propogated EfTect of Cortisob on [3H]Cholisze1izc~rporatiorz cc1B cultures derived from fetal rabfibroblast into Lecithin bit tissues exhibit differing biochemical activiIn contrast to our previous findings with ties and differing responses to the glucocortimixed fetal lung cell cultures (Smith P & al. coid hormone, cortisol, both as a function of 1974; Smith and Torday 1974; Torday et crb. the organ from which they were derived and of B975), cortisol has no effect on the incorporation of LaH]choline into lecithin by confluent gestational age of the material used. Thus, monolayers of fibroblasts from cither the fetal fibroblasts from the fetal lung (whose development may be dependent upon glucocorticoid Iung or skin (p > 0.05) (Table 2 ) . stimulation) (Avcry 1975) convert cortisone C(srki.sol-Corki,~oazeInterconver~~io~~~s to the active hormone, cortisol, this activity As reflected by the metabolism of equirnolar increasing with advancing gestation. The high amounts of EaH]cortisone and [lT]cortissl level of activity in this respect is underscored (Table 31, fibroblasts of fetal lung origin pre- by the demonstrated ability of the cells to con-
CAN. J . PHYSIOL. PHARMACOL. VOI,. 53, 19'15
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TABLE3. Interconversion of [3H]cortisone and [14@]cortisol by fetal rabbit fibroblast cell cultures
Cell line Lung, 20 days Lung, 28 days Skin, 20 days Skin, 28 days
E
+
3.5 15.6 0.5 1.5
Fa (%I
F
f 0.7 f 0.6 1 0.4 k 0.4
0.5 f 8.3 8 . 1 +_ 0 . 0 4 3.2 0.4 3 . 5 L- 0 . 6
OAbbreviations used: E, cortisone; F, bSee
Methods.
+
+
(%I
Net activation" i-3.0 2 0.6 4-15.5 k 0 . 6 -2.7 0.7 -2.0 0.7
+ +
cortisol.
vert the exceedingly high concentrations of the hormone with which they were incubated. This observation could be of significance in view of the predominance of 11-0x0 steroids in the fetal circulation of a number of mammals (Burton and Jeyes 1968; Burton et al. 1970; Mittinger 1974), including man (Hillman and Giroud 1965; Bro-Basmussen et al. 196%). I n contrast, skin fibroblasts do not convert cortisone to cortisol at either gestational age studied. Similarly, near physiological conccntrations of cortisol inodulate the in vitro proliferation of lung fibroblasts in a way which is strongly influenced by gestation; stimulating growth in early gestation and inhibiting growth near term. (The mean cord blood cortisol level at term is 0.5 >( M (Murphy and Diez d'Aux 1972)). On the other hand. the growth of fetal skin fibroblasts is only influenced (negatively at both gestational ages) by cortisol at the highest concentration studied (104 MI. With respect to our previous studies of cortisol action in mixed fetal rabbit lung cell cultures, the present study indicates that some of the responses noted could have been due, in whole or in part, to effects of the hormone on the fibroblast subpopulation. These include stimulation of growth at 20 days (Smith et al. 1974), inhibition of growth at 28 days (Smith et ak. 1974), and the conversion of cortisone to cortisol (Torday et ak. 1975). In contrast, the stimulation of lecithin synthesis, previoglsly in the mixed cultures when exposed to cortisol (Smith et 1974; Smith and Torday 1974; Torday et ak. 1975) was not seen with these fibroblast cultures. Indeed, ultrastructural evidence suggests that, in the mixed changes in type 'I function are responsible for this effect of cortisol: these cells sl~owed increased size and
number of osn~iophilliclamellar bodies and increased rough endoplasmic reticulum in cortisol treated cultures (Smith, B. T., and Bolande, R. P.: unpublished observations). The demonstration of a variety of glucocorticsid effects on cultured fetal lung cells (and, likely, the presence of different glucscorticoidresponsive cell types) makes it obligatory to consider with caution the physiological significance of glucocorticoid receptors detected in homogenates sf whole lung (Giannopoulos 1974; Ballard and Ballard 1974), and underlines the importance of relating receptor characteristics to the biological effects of hormones, as previously demonstrated in the case of enhancement of lecithin synthesis (Torday et ak. 1975). Such considerations likely hold true in othcr steroid target tissues. AVERY,M. E. 1975. Pharmacological approaches to the acceleration of fetal lung maturation. Br. Med. Bull. 31,13-17. BALLARD, P. I,., and BALLARD, R. A. 1974. Cytoplasmic receptor for glucocorticoids in lung of the human fetus and neonate. I. Clin. Invest. 53,477-486. BERLINER, D . , and DOUGHERTY, T. 1961. Hepatic and extrahepatic regulation of coi-ticosteroids.Pharmacol. Rev. 13,329-359. BURTON. A. F., GREENALL, R. M., and TURNELL, R. W. 1970. Corticosteroid metabolism and liver glycogen in fetal and newborn mice. Can. I. Biochem. 4, 178183. BURTON,A . F . , and YEYES, C. L. 1968. Corticosteroid metabolism in fetal and newborn mice. Can. S. Biochem. 46, 15-20. BURTON,K . 1956. Study of conditions and mechanism of diphenylamine reaction for the colorimetric determination of deoxyrihonucleic acid. Biochem. J . 62, 3 15-323. BRO-RASMUSSEN, F . , BUUS,0., and TROLLE, I?. 1962. Ratio of cortisone/cortisol in mother and infant at birth. Acta Endor'rinol.( ~ b h )40,579-583. , FOLCM, J., LEES,M., and SLOANE-STANI~EY, G. H. 1954. A simple method for the isolation and purification of total lipides from animal tissues. I. Biol. @hem. 226, 497-502.
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SMITH AND GIROUD: CORTISOL EFFECTS ON FIBROBLAST CELL CU12TURES
1041
GIANNOPQULOS, G. 1974. Variations in the levels of cytoS. 1972. Metabolism of 4- 14Ctestosterone by serially plasmic glucocorticoid receptors in lungs of various subcultured human skin fibroblasts. J , Clin. Endocrinol. Metab. 34, 133-143. species at different developmental stages. EndocriMURPHY, B. E. P., and DIEZd'Anx, R. 1972. Steroid nology, 94,450458. levels in the human fetus: cortisol and cortisone. J . HILLMAN, D. A., and GIROUD,C. J . P. 1965. Plasma Clin. Endocrinol. Metab. 35,678-683. cortisone and cortisol levels at birth and during the PINSKY, L . , FINKELBERG, R., S'~RAISFIELD, C.. ZILAHI, neonatal period. J. Clin. Endocrinol. Metab. 25, 243-248. B., KALJFMAN, M., and HALL,G. 1972. Testosterone KBTTINGER,G. W. 1974. Feto-maternal production and metabolism by serially subcultured fibroblasts from transfer of cortisol in the Rhesus. Steroids, 23, genital and nongenital skin of individual human donors. Riochem. Biopkys. Res. Commnn. 4.364-369. 229-243. MACIERA-COEI-HQ, A., and PONTEN, J . 1969. Analogy in SMITH,B. T., and TORDAY, J. S. 1974. Factors affecting lecithin synthesis by cultured fetal lung cells. Pediatr. growth between late passage human embryonic and Res. 8,848-85 1 . early passage human adult fibroblasts. J . Cell Biol. 43, 374-387. SMITH,B . T.. TORDAY, J . S., and GIROCD, C. J. P. 1973. MARTIN, G . H.. SPRAGUE, C . A., and EPSTEIN, C . J . 1970. The growth promoting effect of cortisol on human Replicate lifespan of cultured human cells: effect of fetal lung cells. Steroids, 22. 515-524, donor age, tissues, and genotype. Lab. Invest. 23, 1974. Evidence for different gestation-dependent 86-92. ef'fects of cortisol on cultured fetal lung cells. J. Clin. Invest. 53, 1518-1526. MASON,R. J., HUBER,G., and VAUGHAN, M. 1972. SynTORDAY, J . S., SMITH,B. T., and GIRCPIJD, C. J. P. 1975. thesis of dipalmitoyl lecithin by alveolar macrophages. J . Clin. Invest. 51,68-73. The rabbit fetal lung as a glucocorticoid target tissue. MULAY,S., FINKELBERG, R., PINSKY, I-., and SOI~OMCPF:, Endocrinology, 96, 1462-1467.
Effects of Cortisol on Serially Propagated Fibroblast Cell Cultures Derived from the Rabbit Fetal Lung and skin' BARRYT. SMITH'AND C. J. P. ~ J I R O U D ~ Mc (;iil Unierersip, Montreal CImiidren'sHospital Rpseurch Itzstitlate, Montreul, Quebec H31f IP3 Keceived May 20, 1975 SMITH, B. T.. and GIROUD,C. J. P. 1975. Effects of ort ti sol on serially propogated fibroblast cell cultures derived from the rabbit fetal lung and skin. Can. J . Physiol. Pharmacol. 53, 10371041. Corti301 affects the growth of serially propogated, fibroblast cell cultures derived from the rabbit fetal lung in a manner which is dependent upon the gestational age of the material used: early in gestation (20 days), the hormone (10-7-10-5 M) stimulates [6-3Hlthymidine incorporaand 10 M) inhibits this process. tion into DNA, while in late gestation (28 days), cortisol M) at both gestational Cultures derived from the rabbit fetal skin are inhibited by cortisol apes. Fibroblasts derived from lung, but not from skin, efficiently convert cortisone to cortisol and this activity increases with advancing gestation. Cortisol does not affect the incorporation of [7H]cholineinto lecithin by confluent cult~aresof any of the fibroblast types studied.
Introduction The fibroblast is a cell type found in most nlammalian tissues whose main function is considered to be the generation of stromal support. Ht has long been known that fibroblasts cultured in vitru retain, and in many cases reflect, the genetic endowment of their source, allowing for the study and diagnosis of a variety of genetic disorders. Evidence that fibroblasts derived from different organs of the same donor (Martin et al. 1970), or from different anatomical sites on the same organ (Pinsky et al. 1972; Mulay et al. 1972) posscss different functions is more limited. Also, there is only limited evidence to suggest that fibroblasts derived from the same source at different stages of development could exhibit changing in vsro phenotypic expressions (Maciera-Coelho and Ponten 1969; Martin et al. 1970). W e have previously shown that mixed primary cell cultures (including fibroblasts and epithelial elements) from the rabbit fetal lung respond differently to cortisol depending upon the stage of gestation at which they are isolated: early in gestation (20-22 days) the hormone stimulates cellular growth, 'late in gestation (28
days) growth is inhibited while the incorporation of labelled precursors into lecithin is significantly cnhanced (Smith et al. 1974). The present studies were designed to determine whether serially propogated fibroblast cell cultures derived from the fetal lung and skin at different gestational ages (20 and 28 days) would reflect gestation-dependent changes in metabolic activities and responses to cortisol. It was also our aim to determine, by inference, which of the responses to cortisol noted in mixed cultures could be accounted for by the fibroblasts present.
'Supported by the Medical Research Council of Canada. Tellow, Medical Research Council of Canada. Present address, to which reprint requests should be sent: Department of Paediatrics, Queen's University, Kingston, Ontario K7L 3N6. 3Associate, Medical Research Council of Canada.
Biological Pregnant white New Zealand rabbits were obtained from Canadian Breeding Farnas, St. Constant, Que., and killed at 20 o r 28 days gestation with a lethal intravenous dose of pentobarbital. The fetuses were removed immediately to provide tissues for the initiation of cell lines.
Methods Culture media used in this study consisted of Hana's
F 10 medium (Difco, Detroit, Mich.) supplemented with 10% fetal calf serum (Flow Laboratories, Rockville, Md.) and antibiotics (Smith et al. 1974). The followillg radioactive compounds were Furchased from New England Nuclear, Boston, Mass.: [3HH]TdR' (sa 9 Ci/mnaol) , [Me-'Hlcholine chloride (sa 2.34 Ci/mn~ol), [4-'4C~ortisol (sa 55.3 mCi/ mmol ) , and [l ,2-3H]cortisone (sa 40 Ci/rnmol) . Radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrophotometer, model 4322, with an efficiency of 27% for TI under single label counting conditions, and of 9.7% and 48% for 3H and 14C, respectively, under double Babel conditions.
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1038
CAN. I . PHYSHOL,. PHARMACOL. VOL,. 53, 1995
The fetal lungs were removed, washed, minced, and trypsinized into a dispersed cell population as previously described (Smith et ul. 1974). The dispersed, mixed cell populations were placed in 30-mm plastic petri dishes (Falcon). Pure fibroblastic cell cultures, as judged by careful phase contrast microscopy, were obtained simply by changing the media 2 k after implantation, at which time only fibroblasts had adhered to the ciishes (Crystal, R.G.: personal cornm~anicakion). Fetal skin fibroblast cell cultures were initiated from I rnm explants by standard techniques. In ali cases, explants were taken from the upper, anterior abdominal wall just below the xiphisternal junction. The culte~res were maintained in 30-mm plastic petri dishes containing 3 rnB of medium and maintained in an atmosphere s f 5% @OLand high humidity at 37 "C. The media were changed every 48-32 h. The cells were transferred by treatment with buffered trypsin-EDTA solution (containing 0.5 g of trypsin, 1 :250, and 2.0 g of EDTA per litre. Grand Island Biological Co., Grand Island, N . Y . ) with a 1 : 3 split every 7-10 days. All studies to be reported here were carried out between the fourth and sixth passage levels. E,+cr 0f &Tortiso/ on Cell G a o ~ ~ t h The effect of cortisol on cellular growth was determined by the incorporation of [WH]TdR into DNA. Cells were plated at 2 X 10' per dish and grown for 48 h in control media. They were then exposed for 24 h to various concentrations ( lQ-0-10-5 M ) of cortisol in control nmedium, following which they were incubated for 6 h with [SHJTdW (2 pCi) in 3 rnl of serum-free medium. Following the incubation, the media were decanted and the cultures washed with three rinses of ice-cold 0.9% saline. The cells were scraped from the dishes, sonicated in 2 ram1 of saline, and the DNA precipitated by the addition of 2 rani of 20% trichlosoacetic acid. Aliquots of the precipitate were taken for counting and the remainder was used for the determination of DNA content by the diphenylamine colorimetric reaction (Burton 1956), using 2-deoxyribose as standard. Results are expressed as counts per minute of "H per nanomole of DNA. Effect of Cortisol OPT ["HICholine Inc~rjloratiot~ into Lecl'thi~z Cultures were grown to confluence in media containing varying concentrations ( l O - O - I O - W M ) of cortisol. Under these conditions, the DNA content of confluent cultures was not affected by the hormone. The cultures were then incubated for 6 h with serumfree media containing 4 X 10" dpna (1.5 nmsH) of VHIcholine. Following the incubation, the media were dec,mted and the c~mltureswashed with three changes of ice-cold saline. The cellular material Mias then scraped from the dishes in three changes (total 5.0 ml) of methanol. Ten millilitres sf chloroform were added and the n~ixturewas tightly capped and left overnight at room temperature. Five anillilitres of
"Abbreviation : [3H]TdW, 66-%]thymidine.
modified aqueous phase for the Folch extraction (distilled water with 100 mA4 KC1 and 10 mA4 choline) (Folch et ral. 1957; Mason ct a / . 1972) were then added. The mixture was shaken and placed at -20 " C to aid phase separation. The lower (organic) phase was filtered and evaporated to dryness. The lipid extracts were then processed for the chromatographic isolation of lecithin as previously described (Smith et al. 1974). The lecithin fractions were counted for their content of 'Hp and the results expressed as picomoles of ["Hlcholine incorporated into lecithin per dish. Cortisone-Cortisol In terconversioaa,~ The ability of the cells to interconvert cortisone and cortisol ( I l-oxidoreductase activity) was studied by inctabating confluent cultures with a high, equimolar concentration (2 X 18-"1 of [14CCortrtisol (2 >: 10" dpm) and [3H]cortisone ("I 110" dprn). The media were then removed and extracted with three volumes of ethyl acetate. The ethyl acetate extracts were processed for the isolation and purification, by paper chromatography, of cortisol and cartisone as previously described (Smith et aE. 1 9 7 3 ) . The incubated radioactivity (%I and 14C) was always recovered to the extent of 75%. The content of 3H and 'C in the cortisol and cortisone fractions was determined and the percentage conversion of cortisol to cortisone and of cortisone to cortisol was calculated. Since the conversion of cortisol to cortisone results in biological inactivation of the hormone while the reverse results in biological activation (Berliner and Dougherty I961 ), the percentage conversion sf cortisone to cortisol was arbitrarily assigned a positive sign while the percentage conversion of cortisol to cortisone was given a negative sign. The two values were then arithmetically summed to give 'net activation'. That is, positive valiaes indicate the net conversion of inactive to active hormone, while negative values reflect net inactivation. StutisticaI All values reported represent the mean (r+-SD)of triplicate observations. Statistical significance of observed differences was assessed using Student's t test for unpaired data.
Results Eflec-t of Cortisol on Cell Growth Addition of cortisol to the cultbarc media of serially propagated fetal fibroblast cell cultures affects the incorporation of I3H]Tc%R4 illto acidinsoluble material irm a manner which is strongly influenced by gestational agc and organ of origin (Table 1) . Lung fibroblasts are most sensitive to the effects of cortisol: ["HITdR incorporation by 20-day lung fibroblasts is stimulated by cortisol. at cormcentrations as low as 10 - 7 M ( p < 0.005), while this process is i~~hibitcd in the case sf 28-day lung fibroblasts,
3 039
SMITH AND GIMBUD: CORTISOI, EFFECTS ON FIBMBBLAS'T CEI,L CULTURES
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TABLE 1. Effect of cortisol on DNA synthesisa by fetal rabbit fibroblast cell cultures
Cell type Cortisol (M)
Lung (20 days)
Skin (20 days)
Lung (28 days)
Skin (28 days)
OAs measured by the incorporation of [3H]thyrnidine. Each value represents the mean ( + SD) counts per minute of 3H recovered in the acidinsoluble fraction per nanomoles of DNA. bp values for statistically significant diKerences (us. control for each cell type) are sho\sn in parentheses.
TABLE 2. Lack of effect of cortisol on lecithin synthesisa by fetal rabbit fibroblast cell cultures
Cel I type Cortisol (Mb
Lung (20 days)
Lung (28 days)
Skin (20 days)
Skin (28 days)
OEach value represents the mean ( 9 SD) picornoles of [311]choline incorpetrated into lecithin per culture dish.
at doses of IV ( p < 0.OS), and 10--Q:W ( p < 0.02). In the case of fetal rabbit skin fibroblasts, cortisol affects [:%I]TdW incorporation only at the highest concentration studied (10- "V), negatively in cultures from both gestations (20 days, g < 0.02; 28 days, p < 0.05). Further studies will be needed to determine if these effects of cortisol on cell growth depend upon the presence of fetal calf serum.
dominantly reduce cortisone to its active form, cortisol. This activity is significantly greater in cells derived from the 28-day lung as compared to the 20-day lung (15.6 0.6 vs. 3.5 t 44.7, p < 0.001 ) . In contrast, fetal skin fibroblasts predonsinantly oxidize cortisol to cortisone, to a similar extent at both gestational ages.
*
Discussion Our results show that serially propogated EfTect of Cortisob on [3H]Cholisze1izc~rporatiorz cc1B cultures derived from fetal rabfibroblast into Lecithin bit tissues exhibit differing biochemical activiIn contrast to our previous findings with ties and differing responses to the glucocortimixed fetal lung cell cultures (Smith P & al. coid hormone, cortisol, both as a function of 1974; Smith and Torday 1974; Torday et crb. the organ from which they were derived and of B975), cortisol has no effect on the incorporation of LaH]choline into lecithin by confluent gestational age of the material used. Thus, monolayers of fibroblasts from cither the fetal fibroblasts from the fetal lung (whose development may be dependent upon glucocorticoid Iung or skin (p > 0.05) (Table 2 ) . stimulation) (Avcry 1975) convert cortisone C(srki.sol-Corki,~oazeInterconver~~io~~~s to the active hormone, cortisol, this activity As reflected by the metabolism of equirnolar increasing with advancing gestation. The high amounts of EaH]cortisone and [lT]cortissl level of activity in this respect is underscored (Table 31, fibroblasts of fetal lung origin pre- by the demonstrated ability of the cells to con-
CAN. J . PHYSIOL. PHARMACOL. VOI,. 53, 19'15
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TABLE3. Interconversion of [3H]cortisone and [14@]cortisol by fetal rabbit fibroblast cell cultures
Cell line Lung, 20 days Lung, 28 days Skin, 20 days Skin, 28 days
E
+
3.5 15.6 0.5 1.5
Fa (%I
F
f 0.7 f 0.6 1 0.4 k 0.4
0.5 f 8.3 8 . 1 +_ 0 . 0 4 3.2 0.4 3 . 5 L- 0 . 6
OAbbreviations used: E, cortisone; F, bSee
Methods.
+
+
(%I
Net activation" i-3.0 2 0.6 4-15.5 k 0 . 6 -2.7 0.7 -2.0 0.7
+ +
cortisol.
vert the exceedingly high concentrations of the hormone with which they were incubated. This observation could be of significance in view of the predominance of 11-0x0 steroids in the fetal circulation of a number of mammals (Burton and Jeyes 1968; Burton et al. 1970; Mittinger 1974), including man (Hillman and Giroud 1965; Bro-Basmussen et al. 196%). I n contrast, skin fibroblasts do not convert cortisone to cortisol at either gestational age studied. Similarly, near physiological conccntrations of cortisol inodulate the in vitro proliferation of lung fibroblasts in a way which is strongly influenced by gestation; stimulating growth in early gestation and inhibiting growth near term. (The mean cord blood cortisol level at term is 0.5 >( M (Murphy and Diez d'Aux 1972)). On the other hand. the growth of fetal skin fibroblasts is only influenced (negatively at both gestational ages) by cortisol at the highest concentration studied (104 MI. With respect to our previous studies of cortisol action in mixed fetal rabbit lung cell cultures, the present study indicates that some of the responses noted could have been due, in whole or in part, to effects of the hormone on the fibroblast subpopulation. These include stimulation of growth at 20 days (Smith et al. 1974), inhibition of growth at 28 days (Smith et ak. 1974), and the conversion of cortisone to cortisol (Torday et ak. 1975). In contrast, the stimulation of lecithin synthesis, previoglsly in the mixed cultures when exposed to cortisol (Smith et 1974; Smith and Torday 1974; Torday et ak. 1975) was not seen with these fibroblast cultures. Indeed, ultrastructural evidence suggests that, in the mixed changes in type 'I function are responsible for this effect of cortisol: these cells sl~owed increased size and
number of osn~iophilliclamellar bodies and increased rough endoplasmic reticulum in cortisol treated cultures (Smith, B. T., and Bolande, R. P.: unpublished observations). The demonstration of a variety of glucocorticsid effects on cultured fetal lung cells (and, likely, the presence of different glucscorticoidresponsive cell types) makes it obligatory to consider with caution the physiological significance of glucocorticoid receptors detected in homogenates sf whole lung (Giannopoulos 1974; Ballard and Ballard 1974), and underlines the importance of relating receptor characteristics to the biological effects of hormones, as previously demonstrated in the case of enhancement of lecithin synthesis (Torday et ak. 1975). Such considerations likely hold true in othcr steroid target tissues. AVERY,M. E. 1975. Pharmacological approaches to the acceleration of fetal lung maturation. Br. Med. Bull. 31,13-17. BALLARD, P. I,., and BALLARD, R. A. 1974. Cytoplasmic receptor for glucocorticoids in lung of the human fetus and neonate. I. Clin. Invest. 53,477-486. BERLINER, D . , and DOUGHERTY, T. 1961. Hepatic and extrahepatic regulation of coi-ticosteroids.Pharmacol. Rev. 13,329-359. BURTON. A. F., GREENALL, R. M., and TURNELL, R. W. 1970. Corticosteroid metabolism and liver glycogen in fetal and newborn mice. Can. I. Biochem. 4, 178183. BURTON,A . F . , and YEYES, C. L. 1968. Corticosteroid metabolism in fetal and newborn mice. Can. S. Biochem. 46, 15-20. BURTON,K . 1956. Study of conditions and mechanism of diphenylamine reaction for the colorimetric determination of deoxyrihonucleic acid. Biochem. J . 62, 3 15-323. BRO-RASMUSSEN, F . , BUUS,0., and TROLLE, I?. 1962. Ratio of cortisone/cortisol in mother and infant at birth. Acta Endor'rinol.( ~ b h )40,579-583. , FOLCM, J., LEES,M., and SLOANE-STANI~EY, G. H. 1954. A simple method for the isolation and purification of total lipides from animal tissues. I. Biol. @hem. 226, 497-502.
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SMITH AND GIROUD: CORTISOL EFFECTS ON FIBROBLAST CELL CU12TURES
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GIANNOPQULOS, G. 1974. Variations in the levels of cytoS. 1972. Metabolism of 4- 14Ctestosterone by serially plasmic glucocorticoid receptors in lungs of various subcultured human skin fibroblasts. J , Clin. Endocrinol. Metab. 34, 133-143. species at different developmental stages. EndocriMURPHY, B. E. P., and DIEZd'Anx, R. 1972. Steroid nology, 94,450458. levels in the human fetus: cortisol and cortisone. J . HILLMAN, D. A., and GIROUD,C. J . P. 1965. Plasma Clin. Endocrinol. Metab. 35,678-683. cortisone and cortisol levels at birth and during the PINSKY, L . , FINKELBERG, R., S'~RAISFIELD, C.. ZILAHI, neonatal period. J. Clin. Endocrinol. Metab. 25, 243-248. B., KALJFMAN, M., and HALL,G. 1972. Testosterone KBTTINGER,G. W. 1974. Feto-maternal production and metabolism by serially subcultured fibroblasts from transfer of cortisol in the Rhesus. Steroids, 23, genital and nongenital skin of individual human donors. Riochem. Biopkys. Res. Commnn. 4.364-369. 229-243. MACIERA-COEI-HQ, A., and PONTEN, J . 1969. Analogy in SMITH,B. T., and TORDAY, J. S. 1974. Factors affecting lecithin synthesis by cultured fetal lung cells. Pediatr. growth between late passage human embryonic and Res. 8,848-85 1 . early passage human adult fibroblasts. J . Cell Biol. 43, 374-387. SMITH,B . T.. TORDAY, J . S., and GIROCD, C. J. P. 1973. MARTIN, G . H.. SPRAGUE, C . A., and EPSTEIN, C . J . 1970. The growth promoting effect of cortisol on human Replicate lifespan of cultured human cells: effect of fetal lung cells. Steroids, 22. 515-524, donor age, tissues, and genotype. Lab. Invest. 23, 1974. Evidence for different gestation-dependent 86-92. ef'fects of cortisol on cultured fetal lung cells. J. Clin. Invest. 53, 1518-1526. MASON,R. J., HUBER,G., and VAUGHAN, M. 1972. SynTORDAY, J . S., SMITH,B. T., and GIRCPIJD, C. J. P. 1975. thesis of dipalmitoyl lecithin by alveolar macrophages. J . Clin. Invest. 51,68-73. The rabbit fetal lung as a glucocorticoid target tissue. MULAY,S., FINKELBERG, R., PINSKY, I-., and SOI~OMCPF:, Endocrinology, 96, 1462-1467.
